Dendritic spines, the primary targets of excitatory synaptic inputs, are potential sites of both biochemical and structural synaptic plasticity. However, the signal transduction pathways underlying activity-dependent changes in spines are largely unknown. Growing evidence suggests that the Ras-MAPK signaling pathway is one of key signaling pathways for memory formation. This pathway is also thought to be altered by longterm drug abuse and addiction. Using a unique dentate gyrus explant culture system, we recently demonstrated that repeated, spaced membrane depolarizations produce a persistent activation of MAPK signaling and formation of dendritic filopodia and spines that are associated with enduring remodeling of synapses and induction of long-term synaptic plasticity. This project analyzes the spatiotemporal regulation of Ras-MAPK pathway and dendritic spine remodeling after patterned stimulation. We will test the hypothesis: Local patterned activity activates dendritic Ras-MAPK signaling leading to spine remodeling. The first specific aim will test the hypothesis that local stimulation activates Ras. The second specific aim will determine if MAPK is activated by local stimulation. The third specific aim will demonstrate that local repetitive stimulation leads to dendritic remodeling via persistent Ras/MAPK activation. Several innovative approaches are being developed to achieve these aims: 1) methods for localized stimulation, 2) quantitative confocal immunocytochemistry, 3) fluorescence resonance energy transfer (FRET)-based probe to monitor the spatiotemporal activition of the Ras, and 4) confocal time lapse imaging of the dendritic spine remodeling. Our experiments will also provide vital information on whether the Ras-MAPK signaling functions as a synapse-to-nucleus signal and answer the fundamental question of what is the minimal number of synapses required for inducing significant amount of somatic and nucleus MAPK signals. The long-term goal of our research is to delineate the cellular mechanisms and signaling transduction pathways underlying dendritic spine formation and plasticity. The major goal of this Stage I application is to develop the strategies and refine the methodologies for studying intracellular signaling mechanisms downstream to patterned activity, in particular the spatial and temporal aspects of the Ras-MAPK signaling and its role in dendritic spine plasticity. These studies will lay the groundwork to allow us to differentially block dendritic or somatic Ras-MAPK activation and to determine if local Ras-MAPK signaling is necessary for dendritic spine remodeling. The experimental system being developed in this study could be used in the future to evaluate the efficacy of psycho-stimulant drugs for activating this signaling pathway and to identify new therapeutic targets for drug addiction.